This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase IV (hPDE IV) isozymes.
Cyclic nucleotide phosphodiesterases (PDE's) are a family of enzymes that catalyze the degradation of cyclic nucleotides. Cyclic nucleotides, particularly cAMP, are important intracellular second messengers, and PDEs are one cellular component that regulates their concentration. In recent years, five PDE enzymes (PDE I-PDE V), as well as many subtypes of these enzymes, have been defined based on substrate affinity and cofactor requirements (Beavo J A and Reifsnyder D H, Trends Pharmacol. Sci. 11:150 [1990]; Beavo J, in: Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action. Beavo J and Housley M D (Eds.). Wiley: Chichester, pp. 3-15 [1990]).
Theophylline, a general PDE inhibitor, has been widely used in the treatment of asthma. It has been speculated that selective inhibitors of PDE isozymes and their subtypes (particularly the cAMP-specific PDE IV) will lead to more effective therapy with fewer side effects (for reviews, see Wieshaar R E et al., J. Med. Chem., 28:537 [1985] and Giembycz M A, Biochem. Pharm., 43:2041 [1992], Lowe J A and Cheng J B, Drugs of the Future, 17:799-807 [1992]). However, even PDE IV selective drugs such as rolipram suffer from emetic side effects that limit their use. An even more selective approach is to inhibit individual subtypes of PDE IV, each one of which is expected to have its own tissue distribution. If the PDE IV isozyme responsible for efficacy is different from that causing side effects, an isozyme selective drug could separate therapeutic and side effects. The cloning and expression of the human PDE IVs would greatly aid the discovery of isozyme-selective inhibitors by providing purified isoenzymes to incorporate into drug assays.
Mammalian PDE IV, the homologue of the Drosophila Dunce gene (Chen C N et al., Proc. Nat. Acad. Sci. (USA) 83:9313 [1986]), is known to have four isoforms in the rat (Swinnen J V et al., Proc. Nat. Acad. Sci. (USA) 86:5325 [1989]). The cloning of one human isoform of PDE IV from monocytes was reported in 1990 (Livi G P et al., Mol. Cell. Bio., 10:2678 [1990]). From Southern blot data, the authors concluded that this enzyme was probably the only PDE IV gene in humans, with the possible exception of one other isozyme. The same group has recently published the sequence of a second human isoform isolated from brain that they designate hPDE IV-B to distinguish it from the monocyte form, which they designate as hPDE IV-A (McLaughlin M M et al., J. Biol. Chem. 268:6470 [1993]). For clarity, we will use this nomenclature as well.
Our invention relates to the nucleic acid sequences encoding three novel human PDE IV isozymes generated by differential splicing from a single gene. We designate these isoforms as hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. The hPDE IV-B2 sequence encodes a polypeptide nearly identical to that reported for hPDE IV-B (McLaughlin M M et al., J. Biol. Chem. 268:6470 [1993]), and the hPDE IV-B2 splice variant represents the unspliced genomic sequence with respect to the differential splice site. Of the two other splice variants, hPDE IV-B1 encodes the longest polypeptide chain, as well as the N-terminal sequence homologous to its rat homologue, DPD (Colicelli J, et al., Proc. Nat. Acad. Sci. (USA) 86:3599 [1989]).
The novel human PDE IV DNA sequences and their encoded peptides may be used to screen for drugs that are selective for a particular human PDE IV isozyme. Such novel DNA sequences may also be used in assays to detect the presence of a particular PDE IV isozyme in human cell lines, thus providing information regarding the tissue distribution of each isozyme and its biological relevance with respect to particular disease states.
The following abbreviations are used throughout this patent:
BALbronchoalveolar lavagebpbase pair(s)cAMPcyclic adenosine 3′,5′-monophosphatedNTP2′-deoxynucleoside-5′-triphosphatedATP2′-deoxyadenosine-5′-triphosphatedCTP2′-deoxycytidine-5′-triphosphatedGTP2′-deoxyguanidine-5′-triphosphatedTTP2′-deoxythymidine-5′-triphosphatehPDE IV-Ahuman monocyte PDE IVhPDE IV-Bhuman brain PDE IVhPDE IV-B1human brain PDE IV, splice variant 1hPDE IV-B2human brain PDE IV, splice variant 2hPDE IV-B3human brain PDE IV, splice variant 3kbkilobase(s)PCRpolymerase chain reactionPDEcyclic nucleotide phosphodiesterasePDE ICa2+/Calmodulin-dependent PDEPDE IIcGMP stimulated PDEPDE IIIcGMP inhibited PDEPDE IVhigh affinity cAMP-specific PDEPDE VcGMP specific PDERACERapid Amplification of cDNA EndsRTavian myeloblastosis virus (AMV) reverse transcriptaseRT-PCRPCR of RT-transcribed mRNASSC1 × SSC = 0.15 M NaCl, 0.015 Na3 citrate pH 7.0
The nucleotides and amino acids represented in the various sequences contained herein have their usual single letter designations used routinely in the art.